The Campylobacter genus encompasses a large number of morphologically diverse groups (spiral, curved or rod shaped) of bacteria with over 35 species and subspecies reported, 20 of which are found to be either pathogenic to humans causing enteric and extraintestinal illnesses or to colonize a diverse number of sites in humans. (Man, 2011).
Campylobacter jejuni, a Gram negative spiral bacterium, is currently one of the most prevalent food-borne pathogens and a leading cause of bacterial gastroenteritis in humans worldwide. In North America, campylobacteriosis outnumbers the reported cases of illnesses caused by Salmonella, Shigella, Listeria and E. coli combined (Stern & Robach, 2003; Newell et al., 2003; Blaser, 1997). Despite relatively mild diarrheal illness, Campylobacter infection has been associated with severe long-term complications, including: Guillain-Barré Syndrome (GBS), the leading cause of paralysis since the eradication of polio; the non-paralytic variant Miller Fisher syndrome (Humphery et al. 2007; Hariharan et al. 2004; Blaser et al. 1986); reactive arthritis (ReA); and inflammatory bowel disease (IBD) (Rautelin et al. 2000; Gellynck et al. 2008).
It is estimated that between 50-80% of human campylobacteriosis cases can be attributed to chicken consumption, and therefore broiler chicken meat is considered the primary vector for transmitting the pathogen to humans (EFSA Journal 2011; Hermans et al. 2011). Control and reduction of Campylobacter levels in poultry, and chickens in particular, could improve poultry product safety and decrease the incidence of Campylobacter-induced gastroenteritis. Thus, long term complications in humans could be reduced, resulting in saving of tens of billions dollars in hospitalization and other associated costs around the world (EFSA report, 2011; Nyachuba, 2010; Buzby et al. 1997).
Current intervention strategies against C. jejuni and other less frequent strains (e.g., C. coli and C. Ian) are applied at various stages of production, including during poultry breeding (primary interventions), during meat production and/or during meat processing. The most accepted strategy is to prevent Campylobacter spp. from entering the flock by installing hygiene barriers and fly screens, the use of high quality water, reduction of slaughter age and discontinued thinning (Newell & Wagenaar, 2000; Wagenaar et al., 2006, 2008; Lin 2009; EFSA report, 2011). However, the susceptibility of chickens to infection by C. jejuni and its ubiquity in the environment have negatively impacted the success of these biosecurity approaches, highlighting the need for alternative approaches by which the bacterial infection can be controlled or eliminated. A reduction in cecal campylobacter levels of 0.5-5 log10 CFU/g has been reported for administration of bacteriophages to chickens as a feed-additive or veterinary drug (EI-Shibiny et al., 2009; Carrillo et al., 2005; Wagenaar et al., 2005).
More effectively, treatment with bacteriocins added to poultry drinking water completely eliminated the pathogen in 90% of cases or reduced its levels by 106 fold or more (Svetoch et al., 2010). Other biological reagents such as probiotics (Santini et al., 2010; Willis et al., 2008) and plant bioactive compounds (Castillo et al., 2011; Kureckci et al., 2012) have also been used as food or water additives and shown to significantly reduce the campylobacter loads in chicken feces. The bactericidal effects of probiotic strains such as lactic acid bacteria (LAB) against C. jejuni have been attributed to the production of organic acids, bacteriocin or bacteriocin-like substances (Santini et al., 2010; Messaoudi et al., 2011). Many of these approaches have not been widely adopted in the field because of issues such as efficacy, safety, toxicity, scale-up of production and purification, and the development of campylobacter resistance.
Antibiotics such as fluoroquinolone and macrolides have been approved for the treatment of Campylobacter spp. in both poultry and humans. However, their prolonged use in human and animal health has led to a rapid increase of resistant campylobacter strains in many countries around the world and their use is no longer recommended in animal feed stocks (Smith et al., 2010; Luangtongkum et al., 2009; Alfredson et al., 2007; Silva et al., 2011). Medium chain fatty acids (e.g., caprylic acid) and monoacyl glycerols are alternatives to antibiotics and have been used as feed and water additives to control or eliminate the campylobacter loads in chickens (de los Santos et al., 2009; Hermans et al., 2010, Molatova et al., 2010). Nonetheless, data related to the numbers and prevalence of Campylobacter upon treatment with chemical compounds is inconsistent and no clear conclusion could be made on their effectiveness (The EFSA Journal, 2011). Antibiotic therapy including the use of virginiamycin, erythromycin, neomycin or ciprofloxacin to reduce or eliminate the source of infection in poultry or to treat human infection is a useful tool. However, a growing concern regarding wide-spread use of antibiotic treatment in animal production is the development of resistant Campylobacter strains and the fact that antibiotic-resistant Campylobacter from chickens might cause antibiotic-resistant infections in humans.
Vaccination against a large number of infectious diseases is widely used in commercially reared chickens (Clark et al., 2012). Vaccination of poultry for protection against Campylobacter spp. colonization has also been extensively studied. However, identification of a cross-reactive vaccinal target capable of eliciting a rapid and strong immune response over a short period of time (3-4 weeks) coupled with the need for novel adjuvants are some of the challenges to be overcome (de Zoete et al., 2007; Layton et al., 2011; Zeng et al., 2010; Clark et al., 2012). Consequently, no commercial vaccine against C. jejuni is currently available.
A competitive exclusion (CE) approach, first described by Nurmi and Rantala (1973), is based on the establishment of a protective enteric flora using defined or undefined microorganisms from the guts of healthy chickens to prevent campylobacter from occupying its specific niche, especially the cecum (Zhang et al., 2007; Chen, 2001; Stern, 2001). Difficulties in applying the CE approach include a lack of standardization in identifying the complex species in CE products as well as limited and variable success rates in reducing campylobacter infections (Lin, 2009; EFSA Journal 2011).
Lastly, it has been suggested that the susceptibility or resistance of chickens to Campylobacter spp. is dependent on the hosts' genetic system and involves both non-immune and immune mechanisms (Kaiser et al., 2009). Therefore, selective breeding would be a method of choice to expand the genetically inherited resistant chicken lines. In this regard, a 10-100 fold difference in Campylobacter spp. colonization was observed between four inbred chicken lines and the inherited resistance pattern was consistent with single autosomal dominant locus (Boyd et al., 2005). Establishment of resistant chicken lines while preserving meat or egg production and quality, is, however, a time-consuming process with unpredictable results. To date, none of the above-mentioned experimental interventions has been modelled or applied at the field level and, therefore, none has been successfully commercialized.
Antibodies were originally recognized as effective antimicrobial reagents by Behring and Kitasato in the early 1890s (Behring & Kitasato, 1890; Casadevall et al., 2004) and since then, serum therapy became an effective strategy to combat many infectious diseases. The presence of specific antibodies in the serum or intestinal secretions has been associated with resistance of rabbits (Burr et al., 1988; Pavlovskis et al., 1991; Rollwagen et al., 1993) and mice (Dolby & Newell 1986; Rollwagen et al., 1993) to colonization by C. jejuni. In young chickens (less than 2-3 weeks old), the presence of maternal antibodies against Campylobacter spp. delays the onset of colonization and reduces the rate of horizontal spread of C. jejuni in the flock (Sahin et al., 2003), suggesting that passive immunotherapy using anti-Campylobacter spp. antibodies could be an attractive approach for interfering with bacterial colonization in chickens. Indeed, passive immunization with anti-flagella monoclonal antibodies has already been shown to reduce C. jejuni colonization in mice (Ueki et al., 1987). Similarly, the use of hyperimmune anti-C. jejuni rabbit serum or anti-C. jejuni antibodies appear to be effective in diminishing C. jejuni colonization in chickens (Stern et al., 1990). Consistent with this, others have shown that poultry abattoir workers who have high titers of Campylobacter spp.-specific IgGs circulating in their blood rarely acquire campylobacteriosis (Cawthraw et al., 2000). Despite all these observations, antibodies as agents for reducing Campylobacter loads have not gained market attention largely due to the high cost of manufacturing, sensitivity of conventional antibodies to GI tract proteases, lack of effective GI tract delivery systems, and high antigenic variation among Campylobacter spp., which requires multiple antibody preparations to target different strains of Cam pylobacter.
U.S. Pat. No. 8,173,130 (Salzman et al.), U.S. Patent Publication No. 2009/0208506 (Rachamim et al.), and U.S. Patent Publication No. 2010/0239583 (Murthy et al.), describe monoclonal antibodies to flagellin from various Gram-negative bacteria including Campylobacter, which can be used to deter bacterial infection, as well as treat or prevent diseases including inflammatory bowel disease. These antibodies share common disadvantages of such molecules including difficulty in engineering, difficulty in and cost of production, and slow tissue penetration when used in vivo. Additionally, mAb and fragments thereof (for example, scFv and Fab) are very sensitive to GI tract proteases, which is disadvantageous when oral administration is desired.
The presence of specific antibodies in the serum or intestinal secretions of rabbits and mice has been associated with a resistance to gastrointestinal tract colonization by C. jejuni. Studies in chickens also suggest that active immunization can reduce the level of intestinal infection by C. jejuni, but the window of time to obtain a sufficient immune response prior to the early slaughter of chickens, as well as cost and feasibility, make this approach impractical.
Control of Campylobacter at source, particularly within poultry farms, would reduce the risk of human exposure to the pathogen and would have a significant impact on food safety and public health. Advantageously, a safer food supply permits a supplier to avoid costly operational shut-downs and product recalls. Reducing environmental exposure, improving biosecurity, competitive exclusion, vaccination, host genetics selection, and antimicrobial or antibiotic strategies including bacteriophage therapy and bacteriocin treatment are beneficial, but there is still a need for improved strategies to reduce C. jejuni in the food supply. Innovative approaches to the challenges presented by Campylobacter jejuni would be of benefit to the public.
Therefore, there remains a need in the art for a cost-effective method of reducing C. jejuni in the food supply; there also remains a need in the art for antibodies that have high affinity but can overcome the shortcomings of IgGs and their variants.